The method of subtractive hybridization, in which differentially-expressed mRNA species can be identified, will permit the detection of unique gene products. This approach has been used to isolate and begin to identify molecular markers for pancreatic cancers. Initial investigations have focused upon isolation of cDNA from the diploid, near-normal and highly metastatic human pancreatic cell lines obtained from the American Type Culture Collection (ATCC) (HS680.PAN and CAPAN-1, respectively); these cellular materials afford the potential to distinguish genes expressed in pancreatic cancers from those expressed in the normal pancreas. Additionally, these and other cell lines and tissues are being examined for mutations, amplified expression and other altered forms of expression. In particular, gene amplification of known oncogenes, as well as for other novel genes, will be employed as probes. Similarly, molecular examination of genes and their products, particularly between early-stage malignant tissues and highly-metastatic samples by subtractive hybridization, should enable identification of novel, differentially-expressed genes that may be altered compared to their normal cellular counterparts, and more impor- tantly, may identify those genes that may be associated with specific events in this highly-lethal cancer that almost always metastasizes to distal sites. Other molecular procedures, such as GpC methylation studies, PCR assays, and S1-expression protection assays, in combination, will be employed and should enable the detection of specific molecular alterations in specific genes like oncogenes and tumor-suppressor genes, as well as in unique growth-factor/receptor-type genes, or their gene products. Distinctive clones identified in this phase of the investigation are being used to screen and molecularly categorize malignant pancreatic tissue. A cDNA probe will also be used for in situ and confocal laser scanning microscope (CLSM) studies. Improved techniques of this type will be employed to define a variety of new molecular probes that can afford increased sensitivity for detection of early pancreatic neoplasia.